Abstract: The exact etiology of the intellectual disability in Down syndrome (DS) is unknown. Triplication of human chromosome 21 (HSA21) results in widespread changes across the entire epigenome and transcriptome. Recent studies have identified dysregulated gene networks related to oligodendrocyte (OL) development, differentiation, and myelin maintenance in human and mouse DS brains. These transcriptional changes manifest in the delayed onset of myelination, reduced density of myelinated fibers, disruption in their lattice-like organization, and decreased conduction velocity across myelinated fibers, implying that aberrant gliogenesis and white matter alterations in trisomic brains contribute to intellectual deficits in DS. Furthermore, in contrast to the reduction of OLs, an expanded population of astrocytes has been reported in DS. OL cell specification and differentiation is tightly controlled through stage-specific acquisition of defined chromatin marks. Distinct epigenetic signatures, including differing patterns of DNA deacetylation and methylation, and altered histone marks have been observed in DS. Thus, the dysregulation of the genes driving OL development and maturation may be a consequence of aberrant DS-related epigenetic architecture resulting in perturbed OL production and the white matter deficit observed in DS. In our preliminary studies, we differentiated OLs from isogenic trisomic and euploid control induced pluripotent stem cells (iPSCs) derived from people with DS and identified trisomy-related dysregulation of OL commitment, development, and maturation. However, the heterogeneous culture inherent to the published protocol limits downstream analysis and identification of functionally and transcriptionally distinct OL subtypes as well as examination of their developmental trajectories in DS. Thus, our goal is to engineer two isogenic DS-derived iPSC lines to express tdTomato under the control of the endogenous platelet-derived growth factor receptor α (PDGFRα) locus to facilitate the study of the molecular etiology of the white matter deficit in DS. PDGFRα is primarily expressed in proliferating OPCs and PDGFRα+ progenitors, giving rise to mature OLs as well as astrocytes. Thus, DS-derived PDGFRα reporter lines will allow to examine changes in gliogenesis, the imbalance in OL and astrocyte production, and monitor OL development and maturation. Using these lines, we will perform single cell assay for transposase-accessible chromatin (ATAC)-seq to examine how changes in chromatin accessibility affect OL lineage developmental trajectory in DS patient-derived cells. Our studies will conceptually link DS-associated alterations in OL differentiation to the epigenetic architecture as the underlying cause for abnormal OL development leading to the intellectual deficits in DS.

Project narrative: Down syndrome (DS) is caused by triplication of human chromosome 21 and is characterized by intellectual deficits and motor dysfunction, both which may be in part caused by recently described changes in oligodendrocyte (OL) development and abnormal myelin. Based on our preliminary studies on induced pluripotent stem cells derived from people with DS, which show altered OL development in trisomy, we propose to generate isogenic DS patient-derived OL fluorescent reporter lines targeting the endogenous expression of OL progenitor cells marker PDGFRα. With the power our fluorescent isogenic lines will provide, this project will be the first to measure differentiation of trisomic and euploid cell lineages and profile the epigenetic states of oligodendrogenesis at a single-cell resolution to uncover underlying molecular mechanisms of perturbed OL development in DS.